Optical test device for particle counting



June 7, 1966 R. J. GRAMM OPTICAL TEST DEVICE FOR PARTICLE COUNTING Filed Nov. 9, 1962 85.53: tun-m $50 INVENTOR. Ronald J. Gramm BY MIM +5.

HIS ATTORNEYS United States Patent Office 3,254,558 Patented June 7, 1966 3,254,558 OPTICAL TEST DEVICE FOR PARTICLE COUNTING Ronald J. Gramm, Pittsburgh, Pa., assignor to Fisher Scientific Company, Pittsburgh, Pa. Filed Nov. 9, 1962, Ser. No. 236,637 2 Claims. (Cl. 88-14) This application relates to a test device for an electrical impulse counting mechanism. It particularly relates to a test device which forms part of apparatus for measuring a characteristic of a plurality of samples each having the same fixed dimensions but varying in the characteristic being measured. More specifically, it'relates to a test device for a counting mechanism which counts'elec trical impulses generated by particles observed in a plurality of samples, each sample having the same precisely measured volume.

By way of actual example, my test device may be incorporated into apparatus for counting blood cells in which the number of cells present in a fixed and carefully measured sample volume are physically counted, the count of each cell initiating an electrical impulse which is fed to integrating equipment which give-s on a meter the total number of cells present in a given sample volume. My invention will be described with reference to such a blood cell counting apparatus, although it is to be understood that it may be incorporated in other apparatus which produces and counts electrical impulses.

The accompanying drawing is a diagrammatic illustration of blood cell counting apparatus embodying my invention.

Referring to the drawing, a positive displacement pump 1 draws on its suction stroke of blood sample which has been diluted as hereinafter explained from a sample container 2 through a tube 3, a counting chamber 4, a

second tube 5 and discharges it through a waste tube 6 on its exhaust stroke. The diameter of the cylinder of the pump 1 and the stroke of the piston within the pump are carefully controlled so that on each full cycle of the pump a carefully measured sample volume is passed through the counting chamber 4. In a specific embodiment of a blood cell counter, such as is shown in the drawing, an initial presample volume of .23 ml. is drawn through the system to flush it, whereupon the counting system is initiated, and it continues while exactly .5 ml. is drawn through the counting chamber. time, cells in the sample are detected and counted.

After .5 ml. of sample has been drawn through the counting chamber, the counting circuit is cut off and an additional .03 ml. of sample is drawn through the systern before the pump begins its return stroke. On the return stroke, the sample is discharged through the waste tube 6. Thus, every time a count is made, it is made of the actual number of cells present in a precisely measured volume of sample.

A motor driven cam having a plurality of faces, each face having a cooperating cam follower is used to carry out the sequence just described. Referring to the drawing, a motor 7 supplied'by a source of electric power (not shown) drives a cam having cam faces 8, 9, 10 and 11 through a gear reducer 12. A manually operated switch 13, when closed, starts the motor 7 and initiates a counting cycle. A cam' face (not shown) actuates a switch (also not shown) which keeps the motor 7 running after manual switch 13 is actuated by hand and shuts the motor off at the end of the cycle. Rotation of cam face 8 lowers cam follower 8a which permits the lever 14 and spring 15 to lower the piston in the pump 1 and draw the sample through the counting chamber 4. The cam face 9 and cam follower 9a, acting through the lever 16,

During this pinch off the waste tube 6 so as to insure liquid being drawn up through the tubes 3 and 5.

Cam face 10 and cam follower 10a start and end the actual counting period. During the counting of blood cells passing through the counting chamber 4, electrical pulses are generated in a manner hereinafter described and delivered to an amplifier 17, which sends them to a circuit for receiving and counting pulses which will also be hereinafter described. -The cam face 10 and cam follower 10a open and close a switch 18. When this switch is closed, output from the amplifier 17 is grounded and when it is open, pulses from the amplifier pass to the electronic counting mechanism. Therefore, at the start of a counting cycle, the cam face 10 and cam follower 10a open the switch 18 and close it at the end of a counting cycle again grounding the output of the amplifier.

During a counting cycle, pulses from the amplifier 17 pass through a selector switch 19 to a trigger 20, thence to a one shot multivibrator 21. From the multivibrator, the pulses pass through a reed relay '22 to an integrator 23. Cam face 11 and cam follower 11a control a switch 24 which through reed relay 22 controls the input to the integrator. The contacts of switch 24 are closed before switch 18 opens so that the first pulse will be received in the integrator and the contacts of the reed relay 22 open after the contacts of switch 18 close so that the last pulse of the counting period is counted by the integrator. The purpose of the reed relay is to isolate the integrator 23 and meter 25 so as to prevent current leakages back through the circuitry which would cause the meter readings to drift downwardly.

The action of the counting mechanism is such that pulses sent to the amplifier 17 vary'in height and width in accordance with the size of the blood cell actually counted, and the amplifier magnifies these differences. The purpose of the trigger 20 and one shot multivibrator 21 is to shape'the originally irregular pulses into squareshaped waves of equal amplitude and duration with the result that the integrator receives a uniform pulse for each cell counted, and it is possible to calibrate a scale of meter 25 which will read out directly the number of particles represented by the output voltage of the integrator.

The volume of thesample which is observed in the counting chamber 4 is only a little larger than the volume of one of the cells to be counted with the result that substantially'all of the cells pass one at a time through the sensing zone, i.e., the portion of the counting chamber which is actually observed. The blood cells are actually observed by an optical system which employs the principle of black field illumination. Forward scattering of light by particles in a dark field creates flashes of light which strike a light energizable transducer, preferably a photomultiplier tube, which changes the flashes of light into pulses of electrical energy. A lens system 26 focuses light from a light source 27 on the counting zone of the counting chamber 4. A black stop 28 is positioned, be-

tween the light source 27 and the lens system 26, and light from the source lamp passes around the 'black stop 28 and is brought to a focus on the sensing zone of the counting chamber 4 through which the sample and the blood cells pass.

Illuminated particles in the counting chamber are seen againsta dark field. The sensing zone is in the same optical plane as the image of the black stop, and this image and of any particles passing through it is magnified by a lens 29 and ocular 30 and projected onto the light sensitive surface of a photomultiplier tube 31. A conventional power supply and a conventional voltage supply for varying the sensitivity of the tube 31 are used but are not shown in the drawing. Each time a particle enters the sensing zone of the counting chamber, the photomultiplier tube senses a flash of light, converts these flashes of light into electrical pulses of varying duration and intensity and sends them on to the amplifier 17. The pulses are made uniform in height and width as explained above and sent on to the integrator 23.

Blood cell counts are generally expressed as so many red blood cells or so many white blood cells per cubic millimeter of blood. In the operation of the blood cell counter just described, a diluted sample of precisely determined volume is passed through a counting chamber where the number of cells actually present in the sample volume are counted. This actual count multiplied by the dilution factor of the sample, i.e., the number of parts of blood in the number of parts of diluent, gives the blood cell count in terms of cells per unit of volume. If the amount of dilution is maintained'the same, then the meter which is actuated by the output from the integrator can be calibrated to read in red blood cells or white blood cells directly.

The test system which I have invented tests every part of the counter except the optical system by displaying on the meter a counts per unit volume ratio which is obtained when the counter goes through one complete counting cycle with which counts per unit volume obtained in tests of actual samples may be compared. In this manner, the accuracy of counts per unit volume obtained during actual sample tests may be determined.

Referring to the drawing, the test system includes a light energizable transducer 32, preferably a photodiode which, when actuated by light, sends an electric current through the selector switch 19 to the trigger, one shot multivibrator and integrator of the counting circuit. A lens 33 focuses light from the light source 27 onto the photodiode 32. A flicker wheel 34 is mounted on a shaft 35 of the motor 7 and is rotated by the motor. A series of slots are formed in the flicker wheel adjacent its periphery. Theseslots are arranged in a circle about the center of the wheel and are spaced from each other to form a series of alternate light transmitting and opaque segments 36 and 37 about the periphery of the wheel, and

the wheel 34 is positioned so that these segments pass through and interrupt the beam of light passing from the light source 27 to the photodiode 32.

Interruption of the light passing to the photodiode 32 will cause the diode to send a series of pulses to the counting circuit, and the number of pulses received by the circuit will be recorded on the meter 25. It will be noted that the flicker wheel 34 is driven by the same motor which actuates the pump 1. Therefore, by proper selection of the number of segments on the flicker wheel 34, and by proper selection of the gear ratio in the gear reducer 12, there can be sent to the counting circuit the number of pulses which,.when the dilution factor of the sample is taken into consideration, equals the number of counts which in a test of an actual sample would give a full scale reading for white blood cells and a half scale reading for a red cell count. This number can be obtained at will by turning the selector switch 19 to disconnect the amplifier 17 and connect the photodiode 32 to the counting circuit.

An actual operation of the test system will now be de scribed. Using a flicker wheel 34 which has 25 slots through which light reaches the photodiode 32 and a gear ratio of 1,850 to 1 in the gear reducer 12, then 46,250 test pulses are sent out by the photodiode for each full rotation of the cam 8 which actuates the pump 1, and these pulses are admitted to and cut off from the counting circuit by the switch 18, which is actuated by the cam 10. The .5 ml. sample which is observed during an actual sample count is controlled by this same switch as explained above, and, therefore, the number of pulses admitted from the photodiode is directly related to the pump volume. The switch 18 is open to pass pulses to the counting circuit during 225 of rotation of the cam 10, and, therefore, 225/360 46,250 or 28,912 pulses are sent to the counting circuit per .5 ml. of sample. In the actual instrument here described, the bore of the pump 1 and the stroke of the piston in the pump and the actuation of the switch 18 by the cam face 10 were such that the photodiode tube produced 57.8 pulses per cubic millimeter of sample which would be drawn through the counting chamber during an actual count.

In the use of the instrument here described, red blood cell counts and white blood cell counts are made separate- 1y. For these counts a dilution ratio of 62,500 to 1 is used to dilute a blood sample when a red blood cell count is being taken, and a dilution ratio of 250 to 1 is used for a white blood cell count.

In order to obtain a full scale reading on the meter 25, it will be assumed that blood is tested which has a red blood cell count of seven and one-half million red blood cells per cubic millimeter. If this is diluted 62,500 to l, the actual red cell count in the counting chamber will be cells per cubic millimeter, and a white blood cell count diluted as above will produce 60 cells per cubic millimeter. This last ratio is quite close to the 57.8 counts per cubic millimeter ratio which is provided by the test system as described above.

As noted, the sample volume is 500 cubic millimeters and multiplying 500 by 120 (for the actual count obtained from red blood cells) and 60 (for the actual count obtained from white blood cells), we obtain a full scale red blood cell reading of 60,000 and a full scale white blood cell reading of 30,000. A test run on the meter should produce 28,900/60,000 of a full scale for a red blood cell count and 28,900/30,000 of full scale for a white blood cell count.

Thus, the test system employing the flicker wheel and photodiode establishes a proper count per unit volume ratio which may be expected from a count of a blood sample and displays it on the meter. It may be used to check the accuracy and operation of the counting circuitry from the amplifier input to the meter. If the test reading shifts, then the instrument should be adjusted so as to bring the test reading back to its original position. This can be readily done without measuring a sample volume or using an external counter.

While I have described a presently preferred embodiment of my invention, it is to be understood that it may be otherwise variously embodied within the scope of the appended claims.

I claim:

1. A test device for particle counting mechanism comprising A. alight source B. a first light energizable transducer positioned to receive light from said source C. a fixed displacement pump for moving a fixed sample volume containing particles to be counted through a light beam passing from said light source to the transducer, the particles passing through said beam substantially one at a time and the transducer producing an electrical impulse each time a particle passes through the beam,

D. a motor for driving said pump,

B. an electrical circuit to receive electrical impulse and to indicate the number of impulses received,

F. a second transducer energiza-ble by light from a light source,

G. means lfOI periodically interrupting the passage of light from said light source to the second transducer, said means being actuated by the pump motor, and

H. a switch for connecting the first and second transducers alternately to the circuit for receiving electrical impulses and indicating their number whereby the number of impulses produced by the first transducer during movement of the pump motor to move a fixed sample volume can be compared with the number of impulses produced by the second transducer during the same amount of movement of the motor.

2. A test device for particle counting mechanism comprising A. alight source B. a first light energizable transducer positioned to receive light from said source C. a fixed displacement pump for moving a fixed sample volume containing particles to be counted through a light beam passing from said light source to the transducer, the particles passing through said beam substantially one at a time and the transducer producing an electrical impulse each time a particle passes through the beam, I

D. a motor for driving said pump,

E. an electrical circuit to receive electrical impulses and to indicate the number of impulses received 7 F. a second light energizable transducer positioned to receive light from said light source G. a flicker Wheel positioned between the light source and the second transducer and connected to the pump motor to be rotated thereby, and

H. a switch for connecting the first and second transducers alternately to the circuit for receiving elec- =trical impulses and indicating their number whereby the number of impulses produced by the first transducer during movement or the pump motor to move a fixed sample volume can be compared with the number of impulses produced by the second transducer during the same amount of movement of the motor.

References Cited by the Examiner UNITED STATES PATENTS 2,480,312 8/1949 Wolf 250-218 2,773,413 12/1956 Schade 88-14 2,812,686 11/1957 Sinclair 88-14 2,850,239 9/1958 Polanyi et a1 88-14 2,873,388 2/1959 Trurnbo 324-78 2,875,666 3/1959 Parker et a] 250-218 2,920,525 1/1960 Appel et a1. 250-222 2,957,132 10/1960 Burkhart 324-70 2,992,384 7/1961 Malbrain 324-79 2,995,705 8/1961 Walker et al. 324-78 J EWELL H. PEDERSEN, Primary Examiner.

25 ORVILLE B. CHEW 11, Assistant Examiner. 

1. A TEST DEVICE FOR PARTICLE COUNTING MECHANISM COMPRISING A. A LIGHT SOURCE B. A FIRST LIGHT ENERGIZABLE TRANSDUCER POSITIONED TO RECEIVE LIGHT FROM SAID SOURCE C. A FIXED DISPLACEMENT PUMP FOR MOVING A FIXED SAMPLE VOLUME CONTAINING PARTICLES TO BE COUNTED THROUGH A LIGHT BEAM PASSING FROM SAID LIGHT SOURCE TO THE TRANSDUCER, THE PARTICLES PASSING THROUGH SAID BEAM SUBSTANTIALLY ONE AT A TIME AND THE TRANSDUCER PRODUCING AN ELECTRICAL IMPULSE EACH TIME A PARTICLE PASSES THROUGH THE BEAM, D. A MOTOR FOR DRIVING SAID PUMP, E. AN ELECTRICAL CIRCUIT TO RECEIVE ELECTRICAL IMPULSE AND TO INDICATE THE NUMBER OF IMPULSES RECEIVED, F. A SECOND TRANSDUCER ENERGIZABLE BY LIGHT FROM A LIGHT SOURCE, G. MEANS FOR PERIODICALLY INTERRUPTING THE PASSAGE OF LIGHT FROM SAID LIGHT SOURCE TO THE SECOND TRANSDUCER, SAID MEANS BEING ACTUATED BY THE PUMP MOTOR, AND H. A SWITCH FOR CONNECTING THE FIRST AND SECOND TRANSDUCERS ALTERNATELY TO THE CIRCUIT FOR RECEIVING ELECTRICAL IMPULSES AND INDICATING THEIR NUMBER WHEREBY THE NUMBER OF IMPULSES PRODUCED BY THE FIRST TRANSDUCER DURING MOVEMENT OF THE PUMP MOTOR TO MOVE A FIXED SAMPLE VOLUME CAN BE COMPARED WITH THE NUMBER OF IMPULSES PRODUCED BY THE SECOND TRANSDUCER DURING THE SAME AMOUNT OF MOVEMENT OF THE MOTOR. 